Compound marked with tritium, its preparation and its use in the location of nuclear receptors of retinoids

ABSTRACT

6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid marked with tritium; its preparation; and its use as a radioactive marker, in the titration of nuclear receptors of retinoids in the presence of contaminating CRABP, or in the measurement of the affinity of retinoids for these nuclear receptors.

The present invention relates to a new compound, marked with tritium,which is related to retinoids, to its preparation and to its useprincipally in the determination of the affinity of retinoids for theirnuclear receptors and/or in the determination of the cellular content ofnuclear receptors. The peculiarity and advantage of this compound is itsabsence of affinity for the cytosolic binding protein (CRABP).

It is known that retinoids constitute a known class of compounds whichact, in particular, on the proliferation and differentiation of numeroustypes of cells; see, for example, B. A. Pawson et al, Journal ofMedicinal Chemistry, Vol. 25, No. 11, pages 1269-1277 (1982).

Retinoids have been used, in particular, in the treatment of variousdermatological disorders in which an irregularity of the mechanism forcontrol of the proliferation and differentiation of the epidermal cellsis involved; see for example, the work "Update: Dermatology in GeneralMedicine", edited by Thomas B. Fitzpatrick et al (MacGraw-Hill BookCompany), published in 1983, and particularly the chapter by D. B.Windhorst et al, The Retinoids, pages 226-237.

The method of action of retinoids is similar to that of steroids andother effectors interacting with nuclear receptor (Chytil & Ong. Proc.Fed. Am. Soc. Exp. Biol., Vol. 38, 2510-514 (1979). Retinoic acidnuclear receptors (RARs) have been isolated (Daly & Redfern, Eur. J.Biochem., Vol. 168, 133-139, 1987) and the corresponding complementaryDNAs have been cloned and sequenced (Petkovich et al, Nature, Vol. 330,444-450, 1987); (Brand et al., Nature, Vol. 332, 850-853, 1988). Todate, two receptors have been described in man, RAR α (462 residues) andRARβ (448 residues). In addition it has been shown that there exists incytosol a binding protein, known as CRABP (cellular retinoic acidbinding protein), but its role is little known.

The determination of the affinity of a retinoid for the RARs comprises aparticularly interesting method of potential biological evaluation ofsaid retinoid. However, the presence of CRABP can hinder this affinitydetermination for RARs in the case of classic retinoids.

Several experimental methods enable the determination of the affinity ofa ligand for its receptors or binding proteins. In particular, a directmethod can be used if the ligand under consideration is radioactivelymarked, or even by competition with a radioactive ligand if the ligandunder consideration is not radioactively marked.

A good radioactive ligand must, on the one hand, have a high affinityfor its receptors and, on the other hand, have low fixing on any othermolecule. In addition, it must be sufficiently stable to be useful inpractice.

In the case of retinoid acids, the radioactive ligand generally used ismost often tritiated retinoic acid, either in the 2-position (J.Labelled Compounds and Radiopharmaceuticals XVIII, p. 1099 (1980)) or inthe 4-position (German Patent Application No. 3.142.945).

Retinoic acid certainly has an excellent affinity for RARs, with lownon-specific fixing, but the molecule has the disadvantage of becomingrapidly degraded by radiolysis, oxidation and photolysis. On the otherhand, retinoic acid is also capable of being fixed on CRABP.

It has now been discovered that tritiated6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid constitutes a newradioactive ligand which has an excellent affinity for RARs, but has noaffinity for CRABP. In addition, this new ligand is very stable and canbe obtained with high specific activity, which makes it particularlyuseful for the measurement of the affinity of retinoids for RARs andparticularly under conditions where CRABP is also present.

The present invention therefore relates to6-[3(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid marked with tritium,and principally in the 5-position of the methoxyphenyl adamantyl groupand in the 5-position of the naphthoic moiety.

The present invention also principally relates to a new radioactiveligand, such as defined above, having a specific activity greater than10 Ci/mmole (about 370 GBq/mmole) and preferably at least equal to 30Ci/mmole, or about 100 GBq/mmole.

The present invention also relates to a method for the preparation ofthe new radioactive ligand, such as defined above. This method isprincipally characterized by the fact that a multihalogenated alkylester of 6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid isprepared which is then submitted to the action of a tritiated reducingagent, then to the action of a saponification agent.

The halogen, preferably, is bromine.

The reducing agent is, principally, tritium. The reduction reaction iscarried out in accordance with known methods, for example, in thepresence of a catalyst, such as palladium, at ambient temperature.

The tritiated alkyl ester obtained after the reduction is thensaponified in accordance with conventional methods, for example, by analkaline base such as soda, in a methanolic medium.

The halogenated derivative used as the starting product is principally adihalogenated derivative, preferably a brominated derivative. Thedihalogenated derivative is, in particular, the derivative halogenatedin the 5-position of the adamantyl methoxyphenyl group and in the5-position of the naphthoic moiety. The alkyl ester employed is,preferably, a lower alkyl ester (1-6 carbon atoms) such as the methylester.

The multihalogenated alkyl ester of6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid can itself beprepared from the alkyl ester of6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid by the action of ahalogenation agent (in particular a bromination agent). For example, thebrominated derivative can be obtained by the action of bromine indichloromethane at ambient temperature and under a nitrogen atmosphere.

6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid is a known product(hereinafter called Compound I), as well as its esters. It can beobtained in accordance with the process described in European patentapplication No. 86.400785 (199.636).

The present invention also relates to, as an intermediate productobtained in the process described above,6-[3-(1-adamantyl)-4-methoxy-5-halogenophenyl]-5-halogeno-2-naphthoicacid, and in particular the dibrominated derivative.

The present invention also relates to the use of tritiated6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid as a radioactivemarker, principally for the measurement of the affinity of substancessuch as retinoids for RARs, in accordance with a competition method.

The radioactive marker of the present invention can also be used:

in the characterization of antibodies against Compound I, theseantibodies (obtained in accordance with conventional methods forobtaining anti-haptene antibodies) themselves being useful in thedetermination of the amount of Compound I fixed or not on RARs; one canjointly use Compound I and the said antibodies to effect aradioimmunodosage;

in the study of the distribution in vivo as well as cellular andsubcellular of Compound I, without interference with CRABP.

The radioactive marker of the present invention can also be employed inthe study of the mechanism of intracellular action and of the generalmetabolism of Compound I in vivo and in the cells and cellular extracts.

These uses are operated in accordance with conventional methods.

The following non-limiting example illustrates the present invention.

EXAMPLE 1 6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid markedwith tritium

(a) A mixture of the methyl ester corresponding to the cited acid (1 g;2.4 mmoles) and 4.5 ml of bromine in 30 ml of dichloromethane is stirredfor 24 hours.

The mixture is added to 50 ml of water, then extracted withdichloromethane (150 ml).

The organic phase is separated, washed with water, then with a saturatedsolution of sodium bicarbonate and finally with sodium thiosulfate.

It is then dried on anhydrous magnesium sulfate and the solvent isevaporated under reduced pressure.

1.24 g of an orange yellow solid corresponding to a dibrominated productaccording to its mass spectrum are obtained. The position of the bromineatoms is confirmed by analysis of the NMR spectrum of the proton, incomparison with the NMR spectrum of the non-brominated derivative. Theproduct obtained is the methyl ester of6-[3-(1-adamantyl)-4-methoxy-5-bromophenyl]-5-bromo-2-naphthoic acid.

(b) The brominated product obtained is then tritiated in the followingmanner:

10 mg of the dibrominated compound are dissolved in 2 ml of ethanol and1 ml of THF. 17 μl of triethylamine and 14 mg of palladium on charcoal(10%) are added. The medium is stirred for 2 hours under a tritiumatmosphere and the catalyst is removed by filtration. Then the solventis removed by evaporation under reduced pressure. The residue is takenup 3 times with methanol. Thereafter the solvent is removed byevaporation under reduced pressure.

The resulting residue is dissolved in the minimum of tetrahydrofuran andpurified by chromatography on a silica plate, the eluant being a 9:1mixture of dichloromethane and methanol. The fluorescent band under UVradiation at 320 nm is collected.

There is thus obtained the methyl ester of6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid (96 mCi) markedwith tritium in position 5 of the methoxy phenyl adamantyl group and inposition 5 of the naphthoic group.

(c) The preceding tritiated product (96 mCi) is dissolved in 5 ml ofmethanol. 6 μl of 5N soda are added and the resulting mixture is heatedat reflux for 3 hours, cooled, acidified using 2 ml of 6N HCl and thenextracted with 50 ml of ethyl ether.

The ether phase is decanted, washed with water, dried on anhydrousmagnesium sulfate and the solvents evaporated under reduced pressure.The resulting residue is dissolved in 2 ml of ether and purified by highperformance liquid chromatography (eluant: 100% ethyl ether). 83 mCi ofthis product is obtained as a saponification yield of 86%.

The final product is dissolved in 100 ml of methanol. Its purity isverified by thin layer chromatography (silica, above eluant). A singlespot is apparent under UV radiation (254 and 3666 nm) and during betacounting.

By HPLC (inverse phase, ZORBAX ODS column, eluant: 43/36/21/0.2acetonitrile/THF/water/trifluoroacetic acid) a single peak is detectedby UV (320 nm) and by beta detection (continuous liquid scintillation).

The specific activity determined by UV spectrophotometry, then betacounting, is 33.0 Ci/mmole.

The total activity obtained, measured by counting with a liquidscintillator, is 83 mCi.

Characteristics of the Fixing of the Compound of Example 1 on NuclearReceptors RARs

Cultures of F9 cells of murin embryonic carcinoma (normally four 90 mmdishes containing in total 4-7×10⁷ cells per incubation condition) werewashed once with PBS (saline phosphate buffer) and incubated for 3 hoursin a medium, without serum, supplemented with the marked retinoichomolog (to verify the specifity). The cells were then detached with atrypsin-EDTA mixture and numbered with a Burker hemocytometer. Thenucleosol containing RARs was extracted from the purified nuclei usingthe method described by Daly and Redfern (1987) (that is, aftertreatment using DNAse and 0.6M NaCl), and analyzed using highperformance exclusion chromatography (HPSEC). 50 μl of marked extractwere injected on a 9×250 mm GF 250 column (Dupont de Nemours). Theelution was carried out in a buffer 0.3M KH₂ PO₄, pH 7.8 at a rate of 1ml/min. The protein content was followed by measuring the opticaldensity at 280 nm. 28 fractions of 0.3 ml each were collected andcounted in Picofluor (Packard) scintillating agent. For each retinoidconcentration, the number of bonded molecules was determined using thesurface of the radioactivity peak and the calculated concentration ofdemi-saturation (C50). The molecular weight calibration of the columnwas carried out using human albumin (67kDa) ovalbumin (45kDa) andmyoglobin (16.8kDa). The dose-response curves and the competition curveswere analyzed on a computer through non-linear regression using thevarious forms of the Clark equation.

FIG. 1 (A and B) represents the HPSEC profile in the detection ofretinoic acid nuclear receptors (RARs) by the compound of Example 1 in afraction of approximately 45kDa in the nucleosol of F9 cells, incomparison with tritiated retinoic acid.

The F₉ cells incubated with 20 nM of tritiated retinoic acid (specificactivity, 52.5 Ci/mmole) (1A) in the absence (□-□) or in the presence(◯-◯) of 100 times more of cold retinoic acid, or with 20 nM of thetritiated compound of Example 1 (1B) in the absence (□-□) or presence(◯-◯) of 1000 times more of cold compound I. The quantity of ligandlinked to the RARs is expressed in dpm (disintegration/minute).

When the above-described method is used and the F9 cells are incubatedwith the compound of Example 1 at a concentration of 20 nM, a singleradioactivity peak is observed in the nuclear extract (FIG. 1B). Themolecular weight corresponding to the average fraction of the peak isapproximately 45kDa, a number close to the molecular weight of humanRARs, as has been calculated from the sequence of their DNAc (Petkovichet al, 1987; Brand et al, 1988), and is compatible with the 4ssedimentation coefficient of murin receptors (Daly & Redfern), 1987).The fixing is specific since it is abolished when the cells areco-incubated with an excess of 1000 times of the cold compound I (FIG.1B).

On the other hand, by transfection of cellular stock not containingretinoid receptors with expression vectors coding separately for eachretinoid receptor, it is possible to isolate cellular extractscontaining each receptor and to carry out a binding test using theprotocol described above. In this manner, the affinity of the compoundof Example 1 can be determined for each retinoid receptor.

Comparison with the Fixing of Tritiated Retinoic Acid on the NuclearReceptors RARs

On FIG. 1A it can be noted that the radioactive peak elutes at the sameposition as with the compound of Example 1. On the other hand, thisfixing is also inhibited by an excess of 100 times of cold retinoicacid.

If the HPSEC results are standardized as a function of the number ofcells, that is, by expressing them by molecules of ligand fixed to theRARs per cell, the compound of Example 1 and retinoic acid give a peakwith the same surface corresponding to approximately 4,000 molecules percell.

Characteristics of Fixing the Compound of Example 1 on CRABP BindingProtein

The results concerning the fixing of compound I and retinoic acid onCRABP are shown in FIG. 2A and FIG. 2B. The direct fixing of increasingconcentrations of the compound of Example 1 (FIG. 2B, bottom curve) andretinoic acid (FIG. 2A, top curve) to rat testicle cytosol containingCRABP in the absence ( - ) or in the presence of 1000 nM of cold homologligand (□-□) was studied. The difference ( - ) represents the specificbond.

The fixing of the claimed compound on CRABP has been compared to that ofretinoic acid. The total fixing non-specific +specific ( - ) is obtainedby incubation of increasing concentrations (up to a maximum of 125 nM)of the claimed compound with a constant quantity (0.3 ml) of a rattesticle homogenate (CRABP-rich organ). The non-specific fixing of thetritiated compound is determined in parallel, by measuring the fixingwith increasing concentrations of the radioactive ligand in the presenceof a large excess (1 μm) of cold homolog ligand (□-□). An incubationtime of two hours is necessary for the establishment of balancedconditions.

Once the balance is achieved, the tritiated CRABP-ligand complex isseparated from the non-bonded ligand by filtration on a Sephadex G25exclusion gel column (B10-Rad econo columns, 0.5×20 cm): the complex iseluted (elution peak at 2.5 ml), while the non-bonded ligand is retainedin totality on the column. The result is total separation between thebonded and non-bonded product. The elution is done directly inscintillation tubes and the radioactivity is measured by counting.

On FIG. 2B, at the bottom, it can be noted that the compound claimedexhibits no specific fixing on CRABP, the specific fixing curves andnon-specific fixing curves being totally superimposable.

Comparison with Tritiated Retinoic Acid

Under these same conditions, the fixing of retinoic acid on CRABP (FIG.2A, at the top) can be studied. On FIGS. 2A and 2B the abscissarepresents the concentrations of free retinoic acid and the ordinaterepresents the concentrations at equilibrium of the bondedligand-protein complex.

The difference between the total fixing curve ( - ) and non-specificfixing (□-□) gives the specific fixing curve ( - ). Analysis of thiscurve provides the dissociation constant at equilibrium (Kd) which isthe concentration of the product required for 50% saturation of CRABP.This constant is a measurement of the affinity of the ligand for itsbonding protein, Kd being inversely proportional to affinity (thesmaller the Kd, the greater the affinity). This analysis gives forretinoic acid a Kd value of 2 nm. The specific fixing is saturable andreversible. Non-specific fixing is linear and non-saturable in the rangeof concentrations employed, and represents less than 20 percent of thetotal fixing.

What is claimed is:
 1. 6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoicacid marked with tritium.
 2. The tritiated6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid of claim 1, markedin position 5 of the adamantyl methoxyphenyl group and in position 5 ofthe naphthoic moiety.
 3. The tritiated6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid of claim 1 having aspecific activity of at least 370 GBq/mmole.
 4. The tritiated6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid of claim 1 having aspecific activity of at least 1100 GBq/mmole.
 5. In a method, using aradioactive marker, to study in vivo, cellular and subcellulardistribution of 6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid,without interference with CRABP, the improvement comprising, as theradioactive marker, tritiated6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid.
 6. In a method,using a radioactive marker, to study the metabolism of6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid in vivo and incells and cellular extracts, the improvement comprising, as theradioactive marker, tritiated6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid.